Human tissue-type plasminogen activator (tPA) is a key physiological regulator of fibrinolysis. It converts the zymogen plasminogen into plasmin, the enzyme which degrades the fibrin network of the thrombus. Apparently, in the presence of a clot, both tPA and plasminogen bind to fibrin and form a ternary complex in which plasminogen is efficiently activated. The affinity for fibrin makes tPA clot-specific, and useful as a therapeutic agent for fibrinolytic therapy in man. Stump et al., Semin. Thromb. Hemos., Vol. 16, No. 3. pp. 260-273 (1990); Lijnen et al., Fibrinolysis, Vol. 3, pp. 67-77 (1989).
The principal physiological regulator of tPA appears to be a specific, fast-acting, plasminogen activator inhibitor type-1 (PAI-1). PAI-1 is a protein of a molecular weight of about 50,000 which binds to tPA in a 1:1 complex, and inactivates it. Recent clinical studies suggest that elevated levels of PAI-1, by reducing the net endogenous fibrinolytic capacity, may contribute to the pathogenesis of various thrombotic disorders, including myocardial infarction, deep vein thrombosis, and disseminated intravascular coagulation. Van Mourik et al., J. Biol. Chem., Vol. 259, pp. 14914-14921 (1984); Colucci et al., J. Clin. Invest., Vol. 75, pp. 818-824 (1985); Almer et al., Thromb. Research, Vol. 47, pp. 335-339 (1987); Hamsten et al., New England J. of Medicine, Vol. 313, pp. 1557-1563 (1985); Wiman et al., J. Lab. Clin. Med., Vol 105, pp. 265-270 (1985).
Two forms of PAI-1 differing in tPA inhibitory activity and referred to as active and inactive or latent (inactive/latent) forms have been observed with PAI-1 protein from both natural and recombinant sources. Reilly et al., J. Biol. Chem., Vol. 265, No. 16, pp. 9570-9574 (1990); Lambers et al., Fibrinolysis, Vol. 2, Supp. 1, p. 33 (1988); Vaughan et al., J. Clin. Invest., Vol. 84, pp. 586-591 (1989). Considerable efforts have been directed to maximizing the amount of the active PAI-1 protein isolated, and researchers have achieved some success in this regard. For example, it has been reported that inactive/latent PAI-1 protein may be converted to an active form by treatment with denaturants such as sodium dodecylsulfate (SDS), guanidium hydrochloride and urea, or with negatively-charged phospholipids. Reilly et al., J. Biol. Chem., Vol. 265, No. 16, pp. 9570-9574 (1990); Vaughan et al., J. Clin. Invest., Vol. 84, pp. 586-591 (1989). Recombinant techniques have also been developed which yield substantial quantities of functionally active, E. coli-expressed, human PAI-1 protein. Reilly et al., J. Biol. Chem., Vol. 265, No. 16, pp. 9570-9574 (1990); Sisk et al., Gene (Amst.), Vol. 96, pp. 305-309 (1990); Davis et al., U.S. Ser. No. 350,264, filed May 11, 1989, entitled "High Level Expression of Functional Human Plasminogen Activator Inhibitor in E. coli". In addition, purification procedures have been discovered in which active PAI-1 may be separated from the inactive/latent form. Hayman et al., U.S. Ser. No. 671,433, filed Mar. 20, 1991, entitled "Purification of Active and Inactive/Latent Forms of Plasminogen Activator Inhibitor-1". Although some progress has been made in maximizing the amount of active PAI-1 protein, one problem which has not been fully addressed to date is the inherent instability of the PAI-1 active form. Indeed, it has been reported by researchers that active PAI-1 is rapidly converted following synthesis to a latent form by some unknown mechanism. Reilly et al., J. Biol. Chem., Vol. 265, No. 16, pp. 9570-9574 (1990); Kooistra et al., Biochem. J., Vol. 239, pp. 497-503 (1989). In view of the labile nature of the active form, some scientists have concluded that obtaining a pure sample of active PAI-1 is "an unrealistic goal". Franke et al., Biochim. Biophys. Acta, Vol. 1037, pp. 16-23 (1990).
Thus, although some success has been achieved in increasing the amount of active form PAI-1 obtained, means for stabilizing this active form and minimizing its conversion to inactive/latent PAI-1 are needed. The present invention is directed to this important end.